Protein antigens from pathogens have long been used in vaccines, designed to elicit neutralising antibody or cell-mediated immune responses in the recipient, specific for the antigen. Proteins however are generally not good at eliciting certain types of cell-mediated immune response, particularly the generation of effector T-cells (including cytotoxic T-cells), which are a desirable component of the response for a great many vaccines (particularly those directed against intracellular pathogens or cancer antigens). Latterly, vaccines have been developed based on naked DNA, usually plasmid DNA produced from E. coli but containing appropriate promoter sequences for expression in mammalian cells. These latter vaccines have transpired to be good at generating ceil mediated immunity (involving effector T-cells, such as interferon-.gamma. secreting antigen-specific T-cells and antigen-specific cytotoxic T-cells), but are poor at generating antibodies against the encoded and expressed antigen. Antibodies are an important component of the protective immune response for a great many pathogens—particularly bacteria and certain viruses such as the influenza viruses. Various remedies have been proposed and explored to rectify the deficiencies of DNA-based vaccines as described below.
Liposomal formulation has been used to enhance the immunogenicity of vaccine antigens, in the protein form, for many years. Liposomal formulation has also been applied in recent years to the formulation of DNA for vaccine purposes. These are studies which have described the co-formulation of plasmid DNA with proteins using liposomes. However, these studies of liposomal co-formulation of DNA with protein have generally used plasmids encoding immunostimulatory cytokines or other biologically active proteins—other than antigen itself. To incorporate the protein form of the antigen itself into a vaccine composition containing a nucleic acid which is designed to express the protein in vivo would seem unnecessary. We are aware of only one publication which has used protein antigen as an additive in the formulation alongside DNA (Alvarez-Lajonchere, L., et al., Mem Inst Oswaldo Cruz, Rio de Janiero, 97(1):95-99. January 2002). Unlike the present invention, no enhancement of antibody response was seen by these authors in co-formulations of the antigen-encoding DNA and its cognate protein compared to immunizations with the protein alone. The formulations used by Alvarez-Lajonchere, et al., comprised mixtures of the active nucleic acid (a plasmid encoding the core antigen of hepatitis-C virus) plus irrelevant carrier DNA and polyethylene glycol, and the protein. Following injection, the protein and the active DNA (which were not physically associated in the mixture) would diffuse independently and reach antigen presenting cells separately. The negative findings of Alvarez-Lajonchere would suggest, to a person skilled in the art, that formulation of protein with its cognate DNA was not a promising way to achieve improved immune responses, at least not improved antibody responses.
In WO-A-9930733 the immune response to a nucleic acid vaccine is proposed to be enhanced by simultaneous administration of a cognate protein. The two components do not need to be administered in the same composition. Both must merely be administered during the induction phase of the immune response with the protein preferably being masked or held back until after the nucleic acid has printed the immune system. In some examples of vaccine comprised naked DNA and naked protein antigen in physical admixture. In others the protein antigen was formulated for delayed release in a biodegradable polymer-alum formulation admixed with naked DNA.
In WO-A-9728817 vaccines are intended to deliver nucleic acid and protein antigen into antigen presenting cells. The nucleic acid may express the same protein as the protein antigen. The nucleic acid and protein are complexed, e.g., by covalent conjugation. The complex may be formulated as a synthetic virus-like particle. It is also suggested that liposomal systems may be used but there are no examples as to how both protein an dnucleic acid should be incorporated into such systems, nor does the specification include any quantitative results for in vivo tests but predicts results which may not in practice occur, especially class II responses.
It is known that non-coding plasmid DNA has an immuno-adjuvant action when coentrapped with peptides in liposomal vesicles (Gursel, M., et al., Vaccine (1999) 17:1376-1383) and that DNA with CpG motifs has an immuno adjuvant effect on naked DNA and peptide vaccines (Klinman, D. M. et al., Vaccine (1999) 17:19-25).